Comparison Between Numeric Parameters From Acute and Chronic Venous Leg Ulcers: Identification of Potential Objective Nonhealing Parameters

Chronic ulcers are recurrent, affecting 1.0 to 1.3% of the adult population at some time in their life.¹ Approximately 75% of these cases correspond to chronic venous leg ulcers (CVLU).¹

Many factors are involved in the CVLU healing process failure.² Experts generally agree that therapeutic efficacy increases through a multipronged treatment strategy: infection management, biofilm elimination, exudate volume and composition management, growth factor adjustment, hypoxia environmental readjustment, and pain control.^3-5 The World Union of Wound Healing Societies has emphasized that effective wound assessment is critical to establish a diagnosis and to identify any comorbidities or complications that may contribute to a delay in the healing process.⁶ Such broad-based management of patients with CVLUs, however, is usually incomplete, mostly due to varying wound assessment and therapeutic approaches.^7,8

The consensus tools for chronic wound assessment are TIME (tissue, inflammation/infection, moisture imbalance, epithelial edge advancement) and MEASURE (measure, exudates, appearance, suffering, undermining, re-evaluate, edge).^9,10 On the other hand, NERDS (nonhealing, exudate, red and bleeding wound surface, debris, smell) and STONES (size, temperature, os probe, new breakdown, exudate, smell) are mnemonics employed to differentiate colonization and critical infection in chronic wounds by observation.^11,12 Finally, Fletcher proposed an assessment tool that took into consideration a complete clinical and socioeconomic characterization of the patient.¹³ The mentioned protocols are commonly applied in clinics for patient follow-up, allowing the observation of the involution/evolution changes throughout the pathologic course. However, these kinds of clinical assessment are predominantly based on subjective interpretation that depends on the observer’s criteria.¹³ Then, the qualitative nature of the tools leads to inconsistent documentation in the patient’s medical record and may also hinder the interpretation by health personnel, thereby preventing the identification of small changes during ulcer evolution.¹³ The authors are not aware of any studies assessing the efficacy or efficiency of these types of evaluation protocols.

Nevertheless, there are many quantitative clinical parameters that are being used that enable more accurate monitoring of patient ulcers, such as wound surface areas measures,¹⁴ exudate pH,^7,8 wound bed temperature,¹⁵ wound glycemia levels,¹⁶ and wound lactate levels.⁷

The treatment schedule based on a subjective characterization could become a trial-and-error therapy, which may result in longer evolutions and high recurrence rates. Therefore, the aim of this work was to compare numeric parameters from acute ulcers and CVLUs to identify potential objective nonhealing parameters.  

Study design. A prospective study was carried out with 93 outpatients with venous ulcers who attended the Dermatology Service of President Nicolas Avellaneda Hospital between 2016 and 2019. Patients older than 18 years of age with venous ulcers were selected using the following criteria: 1) venous insufficiency diagnosed by venous Doppler ultrasound; and 2) ulcer of any duration or any location with an area measuring from 1 to 250 cm². When patients had more than 1 ulcer, all ulcers that met the inclusion criteria were analyzed. Patients were excluded if there was a history of systemic infection, history of cancer or currently receiving chemotherapy treatment, autoimmune disease, drugs/alcohol abuse, and pregnancy. The cohorts were established according to the previously reported ulcer evolution time^1,2,11 with acute ulcers (evolution of less than 4 weeks) and CVLU (evolution of more than 4 weeks).

Collection of exudate samples. All the samples were collected by the designated investigator. No surgical interventions were performed before the exudate sample collection. Before the data collection and the exudate sampling, conditioning of the wound bed was needed. To do this, the external bandage and dressings were first removed gently so as not to pull on the wound. Before collection, the temperatures of the fluids were measured to avoid inducing changes in the measurements. Then, in situ pH measurements were performed. Afterwards, exudate samples were collected using a syringe, and finally, photographs of the ulcers were taken. At the end of the sampling process, ulcers were cleansed with saline and sterile gauze and then covered with wet gauze and bandage. 

All data were measured using the same instruments and protocols that the hospital used daily. 

Exudate. The exudate pH was determined in situ from measurements (skin pHmeter, Hanna Instruments) taken at the upper end, center, and lower end of each ulcer (Figure 1), and the ulcer average pH was calculated from these 3 values. A syringe without a needle was used to collect exudate from different points of the ulcer. The concentrations of lactate and glucose were measured by spectrophotometric determination using a commercial kit (BioTecnica, Minas Gerais) over aliquots of exudate.

Temperatures. Temperatures of normal skin (T_NS), periulcer zone (hyperpigmented zone surrounding the wound) (T_PU), ulcer edge (T_E), and wound bed average temperature (necrotic, fibrinous and granulation tissue) (T_U) were taken with an infrared thermometer (Flash III, TFA Dostmann). Previous research has demonstrated the temperature patterns among normal skin, wound edge, and wound bed may predict ulcer healing¹⁶; therefore, the authors hypothesized that the difference between ulcer (wound bed) temperature and normal skin temperature or periulcer temperature would be an objective chronicity marker in venous ulcers. For this reason, 2 temperature deltas were calculated: ΔT₁ = [T_NS–T_U] and ΔT₂ = [T_PU–T_U].

Imaging studies. Using reference methodology,^17,18 photographs of ulcers were taken from 15 cm above the affected limb. Total ulcer area and pixel counts corresponding to standardized color range (percentage) of granulation, fibrinous (fibrin or fibrin/biofilm area) and necrotic tissue were determined using Image J v1.50 software (National Institutes of Health).

Bacteriology. In accordance with ethical and clinical guidelines, biopsies were taken from those ulcers in which the process would not compromise healing. To avoid such compromising, patients were excluded if 1) ulcer area <40 cm², 2) there was evidence of re-epithelialization, 3) granulation tissue comprised >60% of the total ulcer area, or 4) signs of severe infection were present. Therefore, only 5 biopsies from acute ulcers (representing 35% of all included patients with acute ulcers) and 13 biopsies from chronic ulcers (representing 16% of patients with CVLUs) were collected. Biopsies were collected using a 3 mm punch from the ulcer edge while the patient was under local anesthesia, and bacterial colonization (colony-forming units per gram [CFU/g] of tissue) was determined (Figure 1). 

Statistics. The statistical significance among the assessment parameters was evaluated using the Kruskal-Wallis t test for nonparametrical data and 1-way analysis of variance for parametrical data. Mean comparison of the data series was evaluated using Student t test and Wilcoxon test for paired data, while the Mann–Whitney U test was used for unpaired data. The analysis was performed using Prism v6.0 (GraphPad Software).

Ethical considerations. This research protocol was approved and monitored by the Independent Medical Ethics Committee from the Argentinean Northwest (CIEM-NOA), San Miguel de Tucumán, Tucumán, Argentina. Patients participated after signing the informed consent document.

Of the 93 patients, 6 had more than 1 ulcer that met the inclusion criteria (up to 2 ulcers in the 6 cases). The number of total ulcers analyzed that met the inclusion criteria was 99.

Evolution time. Based on previous reports, ulcers with less than 4 weeks of evolution time were considered acute.^1,19,20 In this group the evolution time range was 2 to 4 weeks (Table). Consequently, ulcers with more than 4 weeks of evolution time were considered chronic.^1,19,20 In this group, the evolution time range was 8 weeks to 19 years. Approximately 83% of the examined ulcers were chronic, and 49% of the examined ulcers had more than 1 year of evolution.

Exudate. In the whole population, exudates showed a median pH that was alkaline (Table). Seventy-one percent of chronic ulcers and 59% of the acute ulcers had a pH ≥8. Also, there were no significant differences among pH values from different wound bed points in the same ulcer, nor was there a significant difference between the pH of acute and chronic ulcers.

Lactate and glucose. There were no significant differences between lactate and glucose concentrations (mM) measured in acute and chronic exudates (Table).

Temperature. T_PUwas significantly higher than T_NS(P < .05)_, T_E (P < .05) and T_U (P < .001) in acute and chronic ulcers. However, ΔT₁ = [T_NS–T_U] andΔT₂ = [T_PU–T_U] showed no significant differences between acute and chronic ulcers (Table). Fifty- eight percent of the acute ulcers and 59.6% of the chronic ulcers showed a ΔT₁ >1.0ºC. Also, 44% of the acute patients and 63% of the chronic patients showed a ΔT₂ >1ºC.

Imaging. The table shows the ranges of ulcer area and granulation and the fibrinous/biofilm and necrotic tissue percentages in acute and chronic ulcers. There was no correlation between ulcer areas and evolution times, nor were there significant differences in granulation, fibrin/biofilm, and necrotic tissue percentages between acute and chronic ulcers. However, 100% of the acute ulcers and 88% of the chronic ulcers showed <80% of granulation tissue. Also, 50% of the acute ulcers and 46% of the chronic ulcers showed >30% fibrin/biofilm. Furthermore, 93% of the acute ulcers and 91% of the chronic ulcers showed <20% of necrotic tissue. 

Bacteriology. More than 10⁵ organisms per gram of tissue was found in 100% of the analyzed samples from both acute and chronic ulcers. The ranges are shown in the table. 

Wound healing is a complex process that can be affected by changes in pH at each stage.^21,22 During healing, the environment of an acute and chronic wound progresses from an alkaline to neutral and then acidic state.²³ The pH of a chronic wound bed generally exists in the range of 7.15 to 8.9.^22-24 Wounds with a high alkaline pH have been shown to have a lower healing rate and lower healing progression rate compared with those with a pH closer to neutral.^23,24 An alkaline pH also increases the proteolytic activity of matrix metalloproteinase and other proteases, producing destruction of extracellular matrix and growth factors and thereby indirectly preventing healing.²⁴ In contrast, a slightly acid pH prevents infection and allows for greater oxygen release from hemoglobin, causing a better oxygenation.²⁴ More specifically, at a pH of 6.8 to 7.2, the processes of cell multiplication,²⁴ fibroblast proliferation,^24,25 and keratinocyte differentiation, maturation, and proliferation^7,26 are encouraged. 

In the present study, the authors found no significant differences in pH of exudate taken from acute and chronic ulcers. Therefore, to assess whether a wound is progressing toward healing, it is crucial to monitor the pH values of the exudate over time (as several studies have previously recommended).^7,23,24 Acidification of the exudate would be a healing marker independently of wound evolution time. In clinical practice, pH measurements could be performed over any wound site.

Biochemical analysis performed on wound fluid from nonhealing and healing CVLUs showed that fluid glucose levels were 2 to 3 times lower than serum glucose levels.^27,28 In contrast, fluid lactate levels were 2 to 3 times higher than serum lactate levels^27,28 (Figure 2). 

Lactate is an important intermediate of the process of wound repair and regeneration. Notably, healing wounds produce and accumulate lactate, with concentrations sometimes rising to the range of 10 to 15 mM.^7,28 Possibly this increase may be attributed to the increased collagen synthesis and deposition that occurs when lactate concentration rises to 15 mM.^29,30 In venous ulcers, there is a low oxygen concentration caused by a deficient perfusion, alkaline pH, and/or bacterial consumption.^10,25 Consequently, lactate is a by-product from the anaerobic glycolysis.^7,25,32 

However, lactate production is only a minor sequela of hypoxia in wounds.²⁹ It is well established that oxygen levels have a relatively small effect on wound lactate concentration.^30,31,33 While some cells in wounds shift toward lactate production in hypoxia, other cells are heavily reliant on aerobic glycolysis, regardless of oxygen levels.^29,31,34 That is the case of the activated neutrophils that produce lactate and oxidants during respiratory burst. Accordingly, lactate production by neutrophils increases with increased oxygenation, apparently offsetting any decrease in lactate production by fibroblasts, due to the alleviation of hypoxia.^29,34 

In contrast, glucose is a serum metabolite that may appear in exudates due to an exacerbated inflammatory process. A decrease in the exudate glucose concentration may be caused by the bacterial consumption.³⁵ The authors did not find significant differences in lactate and glucose concentrations between acute and chronic exudates (Figure 2). Furthermore, glucose and lactate concentrations were similar to the reported values in exudate from nonhealing wounds²⁷ both acute and chronic. In addition, exudate lactate concentrations were higher than the reference serum values in both acute and chronic ulcers (P < .01) (Figure 2). In addition, exudate glucose concentrations were lower than the serum reference values for both cohorts (P < .001). Considering that glucose is an ultra-filtrating metabolite, these results could indicate a bacterial consumption. 

Previous research has demonstrated that the temperature pattern among normal skin, wound edge, and wound bed would predict healing in chronic wounds.^8,16,23,25 Also, in vitro studies have shown that healing is delayed when T_U< T_NS,^25,36 possibly due to a lack of collagen deposition or to a low fibroblast proliferation.^25,36 All the patients analyzed in this work showed a characteristic temperature pattern among the different evaluated zones. When ΔT₁was analyzed, an ulcer temperature (T_U) that was higher than normal skin temperature (T_NS) could be a sign of intense inflammation sign of the area.^25,37 It is well known that chronic wounds remain in a long-lasting inflammatory phase.³⁷ If inflammation lingers over time, tissue and vascular necrosis will occur, leading the ulcer temperature to drop below skin temperature.³⁸ A similar situation was observed when ΔT₂ was compared with the temperature of the periulcer area. 

In this study, the authors proposed a method to photograph and calculate ulcer area, combined with a previous method to process the imaging and calculate percentages of each type of tissue.^14,18,38,39 The granulation tissue formation was found to be a good evolution marker, while the presence of fibrin, biofilm, and necrotic tissue are indicators of a delayed healing.^39,40 Again, no significant differences between acute and chronic ulcers were found in any of the measured parameters.

Wound healing does not occur when bacterial load is greater than 10⁵ organisms per gram of tissue, as the authors found in the included patients.¹⁰ In the present study, bacterial load was greater than 10⁵ CFU/g of tissue for all analyzed samples in both acute and chronic ulcers. Therefore, considering their microbiology, both groups are assumed to be nonhealing ulcers. 

The authors note the following limitations of this study. First, the low number of biopsy samples obtained may have biased each groups microbiology results. Second, ulcer parameters were assessed only once; therefore, this study this study makes no long-term observations and provides no follow-up data to examine how numerical parameters may change over time in relation to wound healing.

The authors found that some patients had ulcers that had undergone less than 4 weeks of evolution but already demonstrated nonhealing features. Conversely, some patients had ulcers with longer evolution time but were in remission or moving toward healing. The considered cohorts did not show significant differences in the analyzed parameters, such as ulcer size, proportion of tissue types, pH, temperature values, lactate, glucose, and bacterial colonization of tissue. Therefore, based on the results of this comparison, the authors encourage further research into the use of objective measurable parameters to establish cutoff points to differentiate the “chronic state” of an ulcer from a “nonhealing state” regardless of the evolution time.

Use of easily obtainable and low-cost nonhealing parameters, like the ones proposed by the authors, would be a new approach to accurately monitor treatment efficacy and prevent the use of ineffective therapies for long periods.

Nicolas A. Cerusico, PhD¹; Romina Chavez-Jara, PhD¹; Silvana A. Lopez, MD²; Eugenia Sesto Cabral, PhD¹; Aida Ben Altabef, PhD³; and Alberto N. Ramos, PhD¹

¹Instituto de Biotecnología Farmacéutica y Alimentaria (INBIOFAL-CONICET), San Miguel de Tucumán, Argentina
²Servicio de Dermatología, Hospital de Clínicas Presidente Nicolás Avellaneda, San Miguel de Tucumán, Argentina
³Instituto de Química Física (INQUINOA-CONICET), Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, San Miguel de Tucumán, Argentina.

Address all correspondence to: Nicolas A. Cerusico, PhD, Av. Roca Nº1900. T4000CAN; San Miguel de Tucumán, Argentina; tel: +54 381 4856596; email: ncerusico@gmail.com

none disclosed

This work was supported by grants BID 3664 PICT 2014, BID 2530 PICT 2014, and D-TEC 0022/2013 from the National Agency for Scientific and Technological Promotion, Argentina.